The Impact Of VDAC1 On Infectious Bursal Disease Virus Replication

Infectious bursal disease(IBD)is an acute,highly contagious and immunosuppressive poultry disease caused by infectious bursal disease virus(IBDV).Chickens infected with IBDV increases susceptibility to other infectious diseases and the risk of subsequent vaccination failure as well,posing a serious threat to the development of poultry industry in the world.Since the genetic coding capabilities of IBDV is relatively limited,the virus needs to rely on host proteins and signaling pathways to complete its own life cycle.Therefore,thoroughly understanding the interaction between viral protein and host factor plays an important role in revealing the infection and pathogenesis of virus and gives rise to a rational design for safer and more effective vaccines.In this study,iTRAQ was used to screen differentially expressed host proteins after IBDV infection.The results demonstrated that VDAC1 expression was upregulated obviously during IBDV infection.Firstly,RT-PCR and Western Blot were used to examine the expression of VDAC1 during IBDV infection,both of the results showed that VDAC1 expression was upregulated during IBDV infection.Secondly,RNAi-mediated knockdown of VDAC1 decreases viral loads of IBDV-infected cells,the expression levels of viral proteins and relative expression of viral RNA.To investigate possible mechanism of VDAC1 in promoting IBDV replication,flow cytometry was used to detect the effect of VDAC1 on IBDV-induced apoptosis.The results indicated that the percentage of cells undergoing apoptosis,caspase-9 and caspase-3 activities following knockdown of VDAC1 were lower than that in scrambled siRNA control cells.Moreover,we performed immunoprecipitation to identify viral proteins which may interact with VDAC1.The results showed that viral proteins VP2 and VP5 which associated with apoptosis displayed no interaction with VDAC1,while RNP subunit VP1 and VP3 interacted with VDAC1 and their interactions need RNA as bridge.Furthermore,it was proved that both VP3 and VP1 exhibited co-localisation with VDAC1 in the cytoplasm observing by confocal microscopy.Since VP1 and VP3 are important components of IBDV RNP complexes and VP1-VP3 interactions promote polymerase activity,double luciferase reporter gene experiments were performed to detect polymerase activity affected by VDAC1.The results showed that VDAC1 remarkably enhances polymerase activity,and VDAC1 enhances IBDV polymerase activity through its ability to stabilise interactions in RNP complexes.In conclusion,our study revealed the molecular mechanism of VDAC1 in promoting viral replication during IBDV infection,which enriches our understanding of IBDV replication regulation and provides a new perspective for further understanding of the interaction between virus and hosts.